Overall Section Summary/Abstract The overarching goal of our FusOnC2 TEST (Targeting the Ewing Sarcoma Translocation) Center is to develop a new mechanistic understanding of EWS/FLI that will allow for the development of new therapeutic approaches for Ewing sarcoma based on inhibiting EWS/FLI. EWS/FLI is an aberrant oncogenic transcription factor formed by the t(11;22)(q24;q12) chromosomal translocation that is the driver oncoprotein of this tumor. Multiple independent studies demonstrate a critical dependence of Ewing sarcoma cells on EWS/FLI such that blockade of the translocation is associated with a loss of proliferation, loss of cell cycle progression and ultimately reversal of oncogenic transformation. Thus, most investigators believe that inhibition of EWS/FLI in the clinic would be the best way to treat patients with Ewing sarcoma. Traditional chemical design of EWS/FLI inhibitors has proven challenging because the molecule does not contain traditionally ?druggable? pockets. We therefore hypothesize that a deeper scientific understanding of EWS/FLI transcriptional function in Ewing sarcoma will allow for identification of new agents. The FusOnC2 TEST Center will focus on two critical molecular features of EWS/FLI required for its transcriptional and oncogenic function: (i) binding of EWS/FLI to GGAA-microsatellites and related DNA response elements, and (ii) EWS/FLI self-association activities to form ?hubs? that coordinate protein-interactions through low-complexity domains to mediate transcriptional function. Targeting EWS/FLI requires diverse expertise to be focused on this problem. The TEST Center is designed to facilitate both independent and highly-focused studies on critical components of EWS/FLI function, while at the same time interacting with the entire FusOnC2 consortium to share new knowledge, techniques, and expertise that will help facilitate the development of new therapeutic approaches to Ewing sarcoma. We believe that the TEST Center will contribute greatly to the overall success of the FusOnC2 Consortium, and help to change the paradigm for how new therapies are developed for pediatric translocation-associated cancers.